Genetically- and spatially-defined basolateral amygdala neurons control food consumption and social interaction

The basolateral amygdala (BLA) contains discrete neuronal circuits that integrate positive or negative emotional information and drive the appropriate innate and learned behaviors. Whether these circuits consist of genetically-identifiable and anatomically segregated neuron types, is poorly understood. Also, our understanding of the response patterns and behavioral spectra of genetically-identifiable BLA neurons is limited. Here, we classified 11 glutamatergic cell clusters in mouse BLA and found that several of them were anatomically segregated in lateral versus basal amygdala, and anterior versus posterior regions of the BLA. Two of these BLA subpopulations innately responded to valence-specific, whereas one responded to mixed - aversive and social - cues. Positive-valence BLA neurons promoted normal feeding, while mixed selectivity neurons promoted fear learning and social interactions. These findings enhance our understanding of cell type diversity and spatial organization of the BLA and the role of distinct BLA populations in representing valence-specific and mixed stimuli.

(A) UMAP of all BLA cells with different cell types identified by established markers.
(B) Heatmap illustrating expression levels of top 5 marker genes in different cell types.
(D) UMAP of GABAergic neurons with annotated clusters.
(E) Molecular signatures of GABAergic neuronal clusters based on expression of selected marker genes.
(F) Heatmap visualization of pairwise correlation matrix of marker gene expression between clusters of GABAergic BLA neurons.The scale bar indicates Pearson's R.
(G) Representative ISH images from Allen ISH data for some of the final 10 marker genes for glutamatergic clusters.
(H) Representative ISH images from Allen ISH data for genes not selected as marker genes due to their widespread expression in many brain regions (Ndst4, Fbn2), low expression (Deptor), or high expression in other brain regions (Spata13).(H)Average of food consumption in the free-feeding assay during calcium imaging (600s): Lypd1 The correlation of response score (Social vs Footshock) (A) Left: Scheme of free-feeding assay.Right: Total distance travelled in free-feeding assay combined with photoactivation (related to Figure 6C).Lypd1 group; n = 14 (ChR2) and 9 mice  Extended Data Fig. 13

. 1 Supplementary Fig. 1 related to Figure 1 :
GABAergic cell clusters in BLA and marker gene selection for glutamatergic cell clusters.

. 2 Supplementary Fig. 2 related to Figure 1 - 2 :. 3 Supplementary Fig. 3
Workflow of snRNAseq and smFISH.Whole Workflow overview from snRNA seq to smFISH image analysis.(A) Nuclei from BLA tissues were harvested and processed through 10x genomics scRNAseq tools.(B) Fixed BLA coronal sections were iteratively hybridized and imaged with 10 marker gene probes with different fluorescence.(A) and (B) were created with BioRender.comreleased under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (C) Image analysis: Four images from 4 rounds of hybridization were superimposed by landmark and serial strain registration, and subregions in BLA were delineated by reference 1 .Cell segmentation (based on nuclear Rnu6 expression) was created by the HALO software with a given parameter (8 μm around the nucleus to target 12 μm of cell diameter).This follows the assumption that an average BLA glutamatergic neuron has a diameter of 10-13 μm 2,3 .The thresholding fluorescence for positive cells for each gene was performed and quantified using HALO software.After preprocessing, the dataset including the x,y positions of positive cells was transferred to the Python workspace for downstream analysis (e.g., PCA and correlation analysis).Details are explained in Methods.Related to Figure 2B: Representative images showing positive cells for each marker gene in BLA.(A) Density heatmaps of selected gene expressions in BLA.Representative samples of 10 marker genes at 4 positions are shown from anterior (left top) to posterior (right bottom).The colors represent expression levels, from blue to red color indicating the lowest and the highest expression levels (radius: 25um).Scale bar: 500µm, M: medial, L: lateral, D: dorsal, V: ventral.
Pairwise correlation heatmap with hierarchical clustering of marker gene expression across all glutamatergic neurons (snRNAseq data, related to Figure 1) Scale bar indicates Pearson's correlation coefficient.(B) Pairwise correlation heatmap with hierarchical clustering of marker gene expression across eight BLA subregions (smFISH data, related to Figure 2); Boxes in heatmap and lines in dendrogram: manual grouping of closely correlated genes by their spatial expression, red for basal (BA), black for lateral (LA) and 1, 3 for anterior (a) and 2,4 for posterior (p).

Supplementary Fig. 6 related to Figure 3 :Supplementary Fig. 7 related to Figure 4 - 7 :
Workflow to anchor transcriptional clusters directly onto BLA space.(A) The scaled sum of the expression of ten marker genes in all cells per cluster from transcriptomics.(B) Representative data tables explaining how to calculate the correlation coefficient (R) from a single smFISH cell (# 3234 for cl4, # 1936 for cl11, # 5065 for cl2) to assign the best correlated transcriptional cluster.(C) Representative table showing the assignment of three example cells from smFISH to the best correlated transcriptional cluster and the corresponding X, Y position in the BLA.Comparison of Lypd1-, Rspo2-, and Etv1positive cells at the level of transcriptomics and spatial expression.(A) UMAP plots showing Lypd1-, Rspo2-, and Etv1-positive clusters in BLA.(B) Expression of Etv1 and Lypd1 in BLA from anterior (left) to posterior (right).The region highlighted with higher magnification is indicated with a white square (Red: BLA Etv1 , Green: BLA Lypd1 neurons); a cell was assigned positive if the number of puncta was higher than the average number of puncta per cell in the whole BLA section.This was subsequently confirmed by visual inspection by two persons blind to the identity of the probe.For all images: Cells marked by the empty arrow are positive for the Green channel, cells marked by the arrowhead are positive for the Red channel, and cells marked by the filled arrow are positive for both channels.(C) Occupancy graphs showing percentages of positive cells expressing one gene or combining two genes, along the anterior-to-posterior axis with representative confocal images (Left: anterior, Right: posterior).Etv1 in Red, Lypd1 in Green.(D) Expression of Rspo2 and Lypd1 in BLA from anterior (left) to posterior (right) (Red: BLA Rspo2 , Green: BLA Lypd1 neurons).For details see B.(E) Occupancy graphs showing percentages of positive cells expressing one gene or combining two genes, along the anterior-to-posterior axis with representative confocal images (Left: anterior, Right: posterior).Rspo2 in Blue, Lypd1 in Green.(F) Expression of Etv1 and Rspo2 in BLA from anterior (left) to posterior (right) (Red: BLA Etv1 , Green: BLA Rspo2 neurons).For details see B.(G) Occupancy graphs showing percentages of positive cells expressing one gene or combining two genes, along the anterior-to-posterior axis with representative confocal images (Left: anterior, Right: posterior).Rspo2 in Blue, Etv1 in Red.